Small Animals, Big Achievements

Small-animal imaging is integral to a variety of preclinical imaging
applications – with researchers monitoring changes in organs and tissue, for
example, in response to physiological or environmental changes. A host of imaging
modalities have been introduced over the years to address the myriad questions that
inevitably arise.

When imaging animals, investigators often want to see how an organ
is working inside the body. They want to know about the consumption of nutrients,
for instance, and the integrity of the organ. Ex vivo analysis isolates the organ
– or whatever it is the investigator wants to know about – from other
factors that might affect its behavior. In vivo imaging, of course, provides a much
better picture of what is happening inside the body.

Dorsal skin-fold chambers provide an excellent microvascular bed
to study tumor growth and development. Shown are images of one chamber prior to
tumor implant (left), following implant of 2000 GFP-fluorescent U87 glioma cells
to study tumor-induced angiogenesis (center), and one week after implantation of
200k cells to study the effect of the tumor burden on the normal microvascular network
(right). Courtesy of M. Waleed Gaber, Baylor College of Medicine.
M. Waleed Gaber and colleagues – Gaber is an associate professor
at Baylor College of Medicine and co-director of the small-animal-imaging facility
at Texas Children’s Hospital, both in Houston – wanted to know what
factors influence the health of vasculature surrounding tumors in the central nervous
system, with the goal of optimizing the efficacy and safety of anticancer therapies.

Using their own fluorescence microscopy techniques and incorporating
the CoolSNAP and Cascade II cameras from Tucson, Ariz.-based Photometrics, they
imaged long-term changes in the same section of cerebral microvasculature in live
laboratory mice, determining that tumor necrosis factor-alpha is linked to acute
microvascular damage and astrocyte activation following radiotherapy. These findings
would not have been possible using ex vivo sections, Gaber said.

Researchers have developed fluorescence microscopy techniques with which to image long-term
changes in the same section of cerebral microvasculature in mice over time, to
study tumor growth and development. Shown is glioma (U87) vasculature seen through
a mouse cranial window 10 (a) and 12 days (b) posttumor implant. The arrowhead draws
attention to the tortuous vessel structure and rapid change in vasculature in only
two days. Courtesy of M. Waleed Gaber, Baylor College of Medicine.
The approach is called intravital imaging, “which means
you are reaching into the animal and measuring something vital.” In this case,
it gave the researchers access to the microvasculature network and, thus, the blood
supply, either through a window of some sort or through measurements on the surface;
e.g., on an ear, leg or foot.

“The cameras are important because you want video,”
Gaber said, “because you’re measuring something that’s moving
constantly.” The cameras used in the study provided a very high frame rate
as well as very high contrast. The latter was especially important, he added, because
the researchers needed to distinguish between the relatively weak fluorescence signals
and the vascular bed.

In vivo imaging offers a number of advantages, but it also can
present challenges. Investigators might have trouble accessing the organ or area
of interest, for example, depending on where it is. Efficient labeling of the organ
also can be difficult.

In addition, said Rachit Mohindra, associate product manager at
Photometrics, “at extremely low light levels, you have to deal with autofluorescence.”
Tissue tends to autofluoresce as you move closer to the ultraviolet, he said. Researchers
have come up with various means to deal with autofluorescence – trying to
filter it out during image acquisition, for example – “but, really,
the only way around it is to avoid it.”

To this end, there has been a push to develop probes in the near-infrared
range. Fluorescent probes developed for in vivo imaging are typically confined to
wavelengths in the visible range, which can be detected readily and quantitatively
by current CCD technologies. Near-IR probes offer several advantages over these,
however: Biological tissue is more transparent in the near-IR, while autofluorescence
is lowest at these wavelengths.

Photometrics recently released two low-light electron-multiplying
CCD (EMCCD) cameras – the Evolve 128 and Evolve 512 – both engineered
to see clearly into the near-infrared.

The Evolve 128 is a low-light EMCCD camera designed to see clearly
into the near-IR. There has been a push in recent years to develop probes in this
range – biological tissue is more transparent in the near-IR, while autofluorescence
is considerably lower than elsewhere. Courtesy of Photometrics.
The cameras incorporate eXcelon sensor technology, a back-illuminated
CCD and EMCCD detector technology jointly developed by Photometrics, Princeton Instruments
and e2v Technologies. Geared specifically toward biomedical research applications,
the sensor images blue and near-IR wavelengths with higher quantum efficiency and
reduces etaloning, a fringe pattern noise often encountered when imaging in the
near-IR.

Whole-body imaging with 3-D optoacoustic tomography

Because of the strong optical scattering in tissue, all-optical
imaging techniques generally have limited spatial resolution at depths beyond a
few millimeters. At the BiOS Hot Topics session during the Photonics West meeting
in January, Alexander A. Oraevsky of TomoWave Laboratories Inc. in Houston described
how researchers can obtain higher resolution at these depths using optoacoustic
imaging.

The Hot Topics session at the BiOS portion of Photonics West 2011 included a report
of a 3-D optoacoustic tomography system that combines the sensitivity and contrast
of optical imaging and the spatial resolution of acoustic imaging. Shown is a 3-D
optoacoustic volume of a nude mouse. Both kidneys are visualized, as are the spleen
and a partial lobe of the liver. Courtesy of the Journal of Biomedical Optics.
This technique converts the light absorbed in tissue into ultrasound,
which has considerably lower scattering and attenuation in tissue in the frequency
range below 10 MHz. Thus, it offers both the sensitivity and contrast of optical
imaging and the spatial resolution of acoustic imaging.

Oraevsky and colleagues saw the potential of the approach for
small-animal imaging and, in late 2009, while he was working with Fairway Medical
Technologies Inc., the team reported in the Journal of Biomedical Optics a whole-body
three-dimensional optoacoustic tomography system for applications in preclinical
research.

In the system described, either a tissue phantom or a mouse is
placed within a sphere surrounded by a concave arc-shaped array of 64 acoustic transducers.
A pair of pulsed lasers – operating at 755 and 1064 nm, respectively –
illuminate the animal, orthogonally to the array, as it is rotated. The imaging
procedure can be repeated multiple times, Oraevsky said during the Hot Topics session,
creating a virtual array of many thousands of detectors.

Processed images are then projected onto a spherical surface,
producing a 3-D image of the animal. The system can generate images of organs and
blood vessels throughout the body with a spatial resolution of ~0.5 mm.

Optoacoustic tomography is well suited to a range of applications.
Because of the high optical contrast between hemoglobin in the blood and the surrounding
tissue, researchers can use it to visualize blood vessels as well as blood distribution
in organs.

This approach suggests a number of possible uses; for example,
cancer research in which investigators induce primary tumors in internal organs
and observe metastases that spontaneously appear in other parts of the body. Here,
the combination of the sensitivity and contrast of optical imaging and the spatial
resolution afforded by acoustic imaging enables detection of tumors in internal
organs – not just subcutaneous tumors – while the 3-D whole-body-imaging
capabilities allow visualization of both the primary tumors and the associated metastases.

TomoWave is looking into developing optoacoustic contrast agents
involving gold nanorods, which could be used in preclinical imaging as well as in
opto-acoustic ELISA-based immunoassays.